The insulin receptor is unique among tyrosine kinase receptors in that it regulates circulating glucose levels. Proposed studies will analyze unique pathway leading from the insulin receptor to the insulin-responsive glucose transporter (Glut4). Current evidence suggests that insulin receptor phosphorylation of IRS-1, and its SH2 domain-mediated interactions with PI 3-kinase, may be important for Glut4 translocation to the cell surface (where it participates in glucose homeostasis). However, PI 3-kinase is activated by many growth factors, cytokine and antigen receptors that do not regulate glucose homeostasis, and its is important in the trafficking of many proteins, so it is unlikely to act as the unique molecular switch to signal Glut4 translocation. Nevertheless, many alternative insulin receptor substrate and SH2 protein-mediated paths do not regulate Glut4 translocation, including IRS-1 and Shc associated SH2 proteins Grb2, SHPTP2, Nck and Crk. Thus we have been left wondering what triggers Glut4 translocation. We have devised a strategy which uses the insulin receptor kinase and Glut4 translocation as the known initiator and endpoint of this important pathway. Since all known signals emanating from all tyrosine kinase-linked receptors are mediated by SH2 proteins, we assume that an SH2 protein regulates Glut4 translocation. Furthermore, since no known SH2 protein acts as the unique trigger for translocation, we hypothesize that a new SH2 protein acts in this capacity. We have over twelve new SH2 domain proteins in the laboratory, and strategies are proposed to determine which is important in insulin action. Since the major tissues for glucose disposal are muscle and fat, the initial focus was on those five new SH2 proteins showing high-level expression in these tissues. The SH2 domains, cognate peptides and antibodies will be introduced into 3T3-L1 adipocytes to see which blocks glucose transporter translocation. In additional studies the SH2 domains and full-length proteins will be expressed in insulin-responsive 3T3-L1 adipocyte, again looking for effects on glucose transport. These strategies rely on the precise biological endpoint of importance and require no a priori knowledge of particular signaling pathways. Integrated sets of experiments will determine where these new SH2 proteins fit within the known maze of insulin signaling molecules and pathways.